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USING SYNTHETIC APPROCHES TO GLYCOCONJUGATES TO UNDERSTAND CARBOHYDRATE-MEDIATED BIOCHEMICAL INTERACTIONS
Full text linkThis PhD Thesis deals with the synthesis of carbohydrates derivatives as analogues of natural compounds and their biological evaluation as tools for the study of biochemical regulatory pathways.
Carbohydrates, together with proteins, nucleic acids and lipids, are the building blocks on which complex living systems are based. This spectacular class of compounds acts as energetic sources, signaling regulators and components of cell surface. So, they are involved in many biological processes essential for the living cell, as immune response, inflammation, cell growth, cell-cell adhesion and so forth. The possibility to develop biochemical tools based on carbohydrates structures could be used to explain their functional role in many of these mechanisms and how to intervene to conceive new drugs systems for the treatment of several pathologies like cancers and bacterial infections, generally due to altered carbohydrate-mediated interactions.
The topic of this PhD thesis can be divided into two parts. The first one, composed by Chapter 1, 2 and 3, deals with the synthesis of glycosylglycerols ligands of Akt, a protein kinase overexpressed in many cancer cells, while the second part, resumed in Chapter 4, is oriented to the study of the opportunity to employ gold nanoparticles as alternative carriers for saccharide antigens in the vaccinology field.
Protein kinase are enzymes involved in the regulation of many crucial cellular processes like proliferation, differentiation and apoptosis [1]. Among them, the serine/threonine protein kinase B, also known as Akt, plays a key role as a component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis, which is implicated in aberrant tumor cell signaling. Inappropriate activation of the Akt kinase is a common event in human tumors making Akt a critical player in cell survival and, consequently, inhibitors that target Akt are potentially relevant for cancer therapy.
Akt is a soluble cytosolic protein composed of three parts, a N-terminal pleckstrin homology (PH) domain, a central catalytic kinase domain and a C-terminal regulatory domain [2]. The key second messenger phosphatidylinositol 3,4,5 triphosphate [PtdIn(3,4,5)P3 or PIP3], generated by the phosphatidylinositol-3 kinase (PI3K), binds to the PH domain inducing a conformational change of Akt which exposes two site of phosphorylation, specifically threonine 308 and serine 473. Their phosphorylation results in a fully activation of Akt that dissociates from the plasma membrane and can phosphorylate many substrates in the cytoplasm and nucleus [3].
The structure of the phosphatidylinositol PIP3, the natural ligand of Akt PH domain, is composed by an inositol, with a phosphate group in position 3 and a glycerol moiety in position 1 carrying long acyl chains.
Sulfoquinovosylacylglycerol (SQDG) analogues, synthesized in our laboratory, show structural features similar to PIP3, as an acylglycerol portion and a negative head. Furthermore the inositol ring can be easily reconducted to a glucose scaffold in which the pyranosidic oxygen replaces the hydroxyl group in position 2 of the inositol. These likeness prompted us to evaluate them as potential Akt inhibitors both in vitro and in vivo. An ELISA test was performed and the obtained results demonstrated the ability of our compounds to inhibit the Akt. In particular, the presence of the anionic group and a long chain lipophilic portion were necessary for their activity. The most active in vitro compounds were also tested for their antiproliferative activity on cell systems confirming these preliminary results.
Following our interest in the research of potential Akt inhibitor able to bind selectively the PH domain, we decided to further explore the potentiality of different anionic glycolipids. Thus we prepared a new class of compounds with the carboxylate instead of the sulfonate group on the sugar, the glucuronosyldiacylglycerol (GlcADG) analogues.
The Akt inhibitory activity of the GlcADG analogues was evaluated both in vitro and in vivo, indicating again the importance of a negative charge and long acyl chains on the glycerol moiety.
To better understand the action mechanism of Akt inhibition by studying their cell localization, part of this PhD thesis was also directed to the preparation of two fluorescent glucuronides structurally related to the most active GlcADGs. In particular the 7-nitro-1,2,3-benzoxadiazole (NBD) was chosen as the fluorophore and was linked to the glycerol moiety through a N-spacer. The biological evaluation of both compound is now underway.
The second part of this PhD thesis reports the potential application of gold nanoparticles as carrier for saccharide antigens related to Streptococcus pneumoniae. The carbohydrate's world and that of nanotechology combine together seeking to improve the development of fully synthetic carbohydrate vaccines against Streptococcus pneumoniae.
Infection of Streptococcus. pneumoniae is one of major cause of morbidity and mortality in the world, causing pneumonia, bacteremia and meningitis [4].
The S. pneumoniae bacterial cell is surrounded by an outer capsule that consists of structurally diverse polysaccharides (CPSs) which elicit specific antibodies able to confer protection against the bacterium. So, they have been used in the preparation of vaccines, but when polysaccharide are used as antigen, they give a T-independent response and consequently result unable to generate a long immunological memory [5]. Step forward have been made when carbohydrate antigens were conjugate to a protein carrier. Indeed, this conjugation induce T-dependent switch with the production of IgG antibodies and the generation of a long-lasting memory response [6].
However, other problems of saccharide-based vaccines need a solution, e.g. no-specific methods of conjugation of the antigens to the protein carrier, risks of carrier-induced epitope suppression and the process of CPSs isolation and purification from natural sources, which require many checks at different stages raising the cost of the final vaccine. The possibility to use the minimum immunological portion obtained by synthetic methodology can solve this latter, while the search for new carriers might overcome the hurdles encountered with the use of proteins as carrier.
In the last years, gold nanoparticles functionalized with oligosaccharides, named gold glyconanoparticles [7] (GNPs), have been developed in order to mimic the natural presentation of the carbohydrate coating which is present at the cell or pathogen surface. GNPs are characterized to be non-cytotoxic, soluble in biological media and easy to prepare and purify.
Recently, GNPs coated with the synthetic tetrasaccharide repeating unit of Streptococcus pneumoniae serotype 14, identified as the smallest structure necessary to evoke an immunological response [8], OVA323-339 peptide as T helper cell, and -D-glucose to assist water solubility, were prepared. They resulted able to induce IgG antibodies against native polysaccharide of S. Pneumoniae serotype 14, and represent the first example of a fully synthetic carbohydrate vaccine against the bacterium [9].
According to this result, we decided to prepare different GNPs bearing the trisaccharide repeating unit of the 19F serotype of S. pneumoniae, alone or together with the tetrasaccharide related to serotype 14, with the aim to develop new multiantigenic systems displaying different carbohydrate antigens arranged on a single nanoparticle.
As a first step, the trisaccharide repeating unit related to Pn19F and -D-glucose suitably functionalized with a thiol-ending linker were synthesized during the six months spent in the Laboratory of GlycoNanotechology of CIC biomaGUNE center (San Sebastian-Spain). Then, all the GNPs were prepared, characterized for their physical properties and tested in vivo in mice to evaluate their ability to induce specific IgG antibodies against native capsular polysaccharides of S. pneumoniae type 14 and 19F, by Dr. Dodi Safari at Eijkman Institute for Molecular Biology in Jakarta.
Mice were immunized intracutaneously with GNPs in mixture with Quillaja purified saponin as adjuvant. ELISA test with polysaccharide Pn14 and Pn19F as coating material were performed on sera of immunized mice to measure the antibodies against native polysaccharide of Streptococcus pneumoniae. However, IgG able to recognize the native polysaccharide were found only in sera of mice immunized with GNPs carrying the tetrasaccharide Pn14, suggesting that the length of trisaccharide Pn19F is not enough to induce an immune response in vivo. Moreover additional ELISA test using GNPs to cover the plate were also performed [10]. They demonstrated that all the GNPs have been able to induce a specific immune response in vivo, thus demonstrating the antigenicity of the system and confirming the ability of GNPs to act as carrier of saccharide antigens.
1. Cohen, P., Protein kinases — the major drug targets of the twenty-first century? Nat. Rev.Drug Discov. 2002, 1, 309–315.
2. Brazil, D. P., Hemmings, B.A., Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem. Sci. 2001, 26, 657-664.
3. Sale, E. M., Sale, G.J., Protein kinase B: signalling roles and therapeutic targeting. Cell. Mol. Life Sci. 2008, 65, 113-127.4. Bryce, J., Boschi-Pinto, C., Shibuya K., Black, R.E., WHO estimates of the causes of death in children Lancet 2005, 365, 1147-1152.
5. Mond, J. J., Lees, A., Snapper, C.M., T Cell-Independent Antigens Type 2. Annu. Rev. Immunol. 1995, 13, 655-692.
6. Avery, O. T., Goebel, W.F., Chemo-immunological studies on conjugated carbohydrate-protein: The immunological specifity of an antigen prepared by combining the capsular polysaccharide of type III pneumococcus with foreign protein. J. Exp. Med. 1931, 54, 437-447.
7. de la Fuente, J. M., Barrientos, A. G., Rojas, T. C., Rojo, J., Canada, J., Fernàndez, A., Penadés, S., Gold glyconanoparticles as water-soluble polyvalent models to study carbohydrate interaction. Angew. Chem. Int. Ed. Engl 2001, 40, 2258-2261.
8. Safari, D., Dekker, H. A. T., Joosten, J. A. F., Michalik, D., carvalho de Souza, A., Adamo, R., Lahmann, M., Sundgren, A., Oscarson, S., Kamerling, J. P., Snippe, H, Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneuminiae type 14. Infect. Immun. 2008, 76, 4615-4623.
9. Safari, D., Marradi, M., Chiodo, F., Dekker, H. A. T., Shan, Y., Adamo, R., Oscarson, S., Rijkers, G. T., Lahmann, M., Kamerling, J. P., Penadés, S., Snippe, H., Gold nanoparticles as carriers for a synthetic Streptococcus pneuminiae type 14 conjugate vaccine. Nanomedicine 2012, 651-662.
10. Chiodo, F., Marradi, M., Tefsen, B., Snippe, H., van Die, I., Penadés, S., High Sensitive Detection of Carbohydrate Binding Proteins in an ELISA-Solid Phase Assay Based on Multivalent Glyconanoparticles. PLoS ONE 2013, 8, 1-11
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The capsular polysaccharide Vi from Salmonella typhi: synthesis and molecular dynamic simulations of short analogue fragments
No full textVi capsular polysaccharide (Vi antigen) is the virulence factor of Salmonella enterica serovar typhi, the causative agent of typhoid fever in humans. It is a linear homopolymer made up of alpha-(1→4)-linked N-acetyl galactosaminuronic acid with a 60-70% O-acetylation at C-3. Vaccination with purified Vi antigen from Salmonella typhi can protect against typhoid fever, although many aspects of the mechanism of action have yet to be established. It has been demonstrated that the immunogenicity of Vi is strongly related to the content of O-acetyl groups and seems not related to the presence of the carboxyl groups. In fact, the acetate groups dominate the molecular surface of the polysaccharide and confer hydrophobic properties to it, probably shielding the carboxylic groups from interaction with other molecules, even if only partial 3-O-acetylation seems necessary to maintain the flexibility of the molecules. Being interested in the study of the role of the negative charge of the Vi biopolymer on the biological activity, we have planned the preparation of analogues where the carboxylic group has been substituted with a pH-independent ionizable group, i.e. the sulfate group. The sulfate group has been selected after preliminary investigations through molecular dynamics simulations on a hexasaccharide Vi antigen fragment, that showed similarities between the conformational behavior of the natural antigen and the sulfate analogue, where the galacturonic residues have been replaced with 6-O-sulfo-galactoses.
Herein we will report our results on the conformational analysis and the stereoselective synthesis of Vi antigen sulfated-analogue fragments
New anionic glycoglicerolipids targeting protein kinase B (Akt)
No full textProtein kinases are enzymes involved in the regulation of many crucial cellular processes like proliferation, differentiation and apoptosis. Among them, the serine/threonine protein kinase B (PKB), also known as Akt, plays a key role as a component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis, which is implicated in aberrant tumor cell signaling. Inappropriate activation of the Akt kinase is a common event in human tumors and Akt is a critical player in cell survival. Thus, inhibitors that target PI3Ks and its downstream effectors, including PKB are potentially relevant for cancer therapy. PI3K activation generates 3-phosphorylated phosphatidylinositols (PI3P) that bind PKB pleckstrin homology (PH) domain promoting PKB activation through its translocation from the cytosol to the plasma membrane, conformational change and final phosphorylation. Thus, inhibitors that target PI3Ks and its downstream effectors, including Akt are potentially relevant for cancer therapy.1
New sulfoquinovosylacylglycerols (SQAG) analogues 12-4 are currently investigated as potential Akt inhibitors, their structure being easily reconducted to PI3P. Here, the synthesis of new anionic glycoglycerolipids 2 derived from the sulfoglycolipids 1 as PI3P analogues targeting the PKB PH domain will be reported. In particular, a series of analogues of natural SQAG in which glucose is -linked to the 2 position of an acylglycerol and a carboxyl replaces the sulfonate group, together with some simpler related -glucuronides, will be shown
A 2,3-carbamate-bearing allyl galactosamine donor for the synthesis of repeating alfa-(1 4)-linked galactosamine units
No full textThe 1,2-cis-linked 2-amino-2-deoxy sugar structure is found in various oligosaccharides of biological importance. The repeating GalpNAc--(1→4)-GalpNAc unit constitutes for example an essential motif incorporated in a range of oligosaccharides, e.g. the repeating unit of the O-antigen moiety of the lipopolysaccharide from Escherichia Coli O142 or Sphaerotilus natans.[1] Vi antigen is instead a linear homopolymer of -(1→4)-linked N-acetyl galactosaminuronic monomers, with a variable degree of O-acetylation at the C3 position. Vi antigen is a capsular polysaccharide found mainly in Salmonella typhi and Salmonella paratyphi C, two serotypes of Salmonella that are responsible for severe infection in humans.[2]
Although in the past years there has been much progress in carbohydrate chemistry, 1,2-cis-selective glycosylation of gluco- or galactosamines is still a challenge. 2-Azidoglycosyl donors, developed many years ago, are still employed for the synthesis of 2-amino-2-deoxy -glycosides, even if glycosylations suffer of low selectivity. Progress in resolving these issues has been made with the development of donors carrying a 2,3-trans-carbamate group, which has attracted much attention as a stereodirecting protection in glycosylation reactions. The fused carbamate ring proved to be a non-participating group and favors the formation of -glycosides. So far, different studies have demonstrated that 2,3-oxazolidinone protected thioglycosides are highly efficient substrates for the synthesis of -linked glycosides.[3]
Herein we report a new 2,3-oxazolidinone protected galactosamine donor, bearing an allyl group at the anomeric position. The allyl group, in addition to its traditional role as a valuable anomeric protecting group, can also be converted into a good leaving group for glycosylation. Thus, the allyl glycoside is first isomerized to the corresponding prop-1-enyl glycoside, which, after chemoselective activation of the anomeric enol ether moiety with a suitable electrophile in the presence of the glycosyl acceptor, leads to the formation of the disaccharide product.[4] This method has the advantage that prop-1-enyl glycoside can be directly derived from allyl glycoside with a variety of facile and highly effective isomerization methods and immediately subjected to glycosylation.
Herein we describe the synthesis of a galactosamine building block, which has the anomeric position protected by an allyl group, and positions 2 and 3 involved in the formation of an oxazolidinone ring. The proper donor and acceptor to perform a -(1→4) glycosylation have been obtained from this common building block. The new 2,3-oxazolidinone protected allyl galactosamine donor has been subjected to glycosylation reactions to study its reactivity and the stereoselectivity of the process.
1. a) Landersjö, C.; Widmalm, G. Biopolymers 2002, 64,283; b) Takeda, M.; Nakamori, T.; Hatta, M.; Yamada, H.; Koizumi,
J.I. Int. J. Biol. Macromol. 2003, 33, 245
2. a) Daniels, E.M.; Schneerson, R.; Egan, W.M.; Szu, S.C.; Robbins, J.B. Infect. Immun. 1989, 57, 31592; b) Heyns, K.;
Kiessling, G. Carbohydr. Res. 1967, 3, 340
3. See for examples: a) Kerns R.J., Zha C., Benakli K.,. Liang Y.Z, Tetrahedron Lett. 2003, 44, 8069; b) Olsson, J. D. M.;
Eriksson, L.; Lahmann, M.; Oscarson, S. J. Org. Chem: 2008, 73, 7181; c) Yang, L.; Ye, X-S. Carbohydr. Res. 2010, 345,
1713; d) Yang, L.; Zhu, J. ; Zheng, X-J. ; Tai, G. ; Ye, X-S. Chem. Eur. J. 2011, 17, 14518
4. Wang, Y.; Zhang, X.; Wang, P. Org. Biomol. Chem. 2010, 8, 4322 and references herein reported.
We acknowledge MIUR-Italy (PRIN 2008) for financial support
Synthesis and molecular dynamic simulations of a Vi antigen sulfated analogue
No full textVi capsular polysaccharide (Vi antigen) was first identified as the virulence factor of Salmonella enterica serovar typhi, the causative agent of typhoid fever in humans. It is a linear homopolymer made up of alpha-(1→4)-linked N-acetyl galactosaminuronic acid with a 60-70% O-acetylation at C-3. This antigen is highly immunogenic and has been used for the formulation of one of the currently available vaccines against typhoid.1 In the context of vaccine development, the search for carbohydrate analogues is stimulated by the intrinsic instability of several polysaccharide vaccines from natural sources or by the need to understand/control the immunological events under analysis.
We are interested in the preparation of oligomeric sulfated analogues of Vi antigen, i.e. analogues in which the 6-carboxylic group has been substituted with a sulfate, to study the influence of the sulfo pH-independent ionizable group on the biological activity. The modification from galacturonic to 6-O-sulfogalactose does not actually generate an isosteric analogue, but in principle should not significantly interfere with the immunostimulating activity of Vi antigen. Indeed, it has been demonstrated that the immunogenicity of Vi is strongly related to the content of O-acetyl groups and not to the presence of the carboxyl group.2 In fact, the acetate groups confer the hydrophobic properties to the molecular surface and dominate the surface of the polysaccharide, probably shielding the carboxyls from interaction with other molecules, even if only partial 3-O-acetylation seems necessary to maintain the flexibility of the molecules.3 So, the modification of position 6 should not influence the biological activity provided that the conformational properties at the alpha-(1→4) glycosidic linkage, as well as the overall geometry of the polymer, are maintained.
Herein we will report the conformational analysis and a stereoselective synthesis of a Vi antigen sulfated-analogue dimeric fragment
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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